PCR, qPCR, and RT-PCR are widely used molecular biology techniques for amplifying and detecting genetic material. Although their names are similar, they differ in the type of template they use, how amplification is monitored, and the instruments required.
The most important distinction is that PCR amplifies DNA, qPCR measures DNA amplification in real time, and RT-PCR begins with RNA and converts it into complementary DNA (cDNA). When reverse transcription is combined with real-time quantification, the method is called RT-qPCR.
What Is PCR?
PCR, or polymerase chain reaction, is a laboratory method used to make millions of copies of a specific DNA sequence.
PCR uses repeated temperature cycles to separate DNA strands, attach primers, and synthesize new DNA. The basic cycle consists of:
- Denaturation: Double-stranded DNA separates at a high temperature, usually around 94–98°C.
- Annealing: Primers bind to complementary target sequences at a lower temperature.
- Extension: A heat-stable DNA polymerase synthesizes new DNA, commonly at approximately 72°C.
After 25–40 cycles, the target DNA is amplified sufficiently for detection.
Main applications of PCR
- Detection of known DNA sequences.
- Cloning and sequencing.
- Genotyping.
- Identification of microorganisms.
- Mutation analysis.
- Forensic DNA testing.
- Research involving genes and plasmids.
Instruments used for PCR
The main instrument is a conventional thermal cycler, also called a PCR machine. It controls the temperature changes required during each PCR cycle.
Common conventional thermal cyclers include:
- Applied Biosystems Veriti Thermal Cycler.
- Bio-Rad T100 Thermal Cycler.
- Bio-Rad C1000 Touch Thermal Cycler.
- Eppendorf Mastercycler.
- Thermo Scientific ProFlex PCR System.
- Agilent SureCycler.
After amplification, the PCR product is usually analyzed using agarose gel electrophoresis. A gel electrophoresis system, power supply, DNA stain, and UV or blue-light transilluminator may therefore also be needed.
What Is qPCR?
qPCR means quantitative PCR, also called real-time PCR. It amplifies DNA while simultaneously measuring the amount of product formed during each cycle.
Unlike conventional PCR, qPCR does not normally require gel electrophoresis to determine whether amplification occurred. The instrument detects fluorescence produced by the accumulating PCR product.
Two common fluorescence chemistries are:
- DNA-binding dyes, such as SYBR Green.
- Sequence-specific fluorescent probes, such as TaqMan probes.
The fluorescence increases as the target DNA is amplified. The cycle at which fluorescence crosses a defined threshold is called the Ct or Cq value. A lower Ct generally indicates a greater amount of starting target nucleic acid.
Main applications of qPCR
- Quantification of DNA.
- Microbial load measurement.
- Copy-number analysis.
- Genotyping.
- Detection of pathogens.
- GMO testing.
- Measurement of transgene levels.
- Quantitative analysis of DNA samples.
Instruments used for qPCR
qPCR requires a real-time PCR instrument. This equipment combines:
- A thermal cycler.
- A fluorescence excitation system.
- Optical detectors.
- Data-analysis software.
- A reaction block for tubes or plates.
Examples include:
- Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems.
- Applied Biosystems QuantStudio 3, 5, 6, and 7 systems.
- Bio-Rad CFX96 and CFX Opus 96 systems.
- Roche LightCycler 96 and LightCycler 480.
- Qiagen Rotor-Gene Q.
- Agilent AriaMx and Mx3005P.
- Mic qPCR Cycler.
The Applied Biosystems 7500 Fast Dx and QuantStudio Dx platforms have been used in real-time RT-PCR diagnostic assays described in CDC and FDA documents.
What Is RT-PCR?
RT-PCR stands for reverse transcription PCR. It is used when the starting material is RNA, rather than DNA.
Because standard DNA polymerase cannot directly amplify RNA, the RNA is first converted into complementary DNA, or cDNA, using an enzyme called reverse transcriptase. The cDNA is then amplified using conventional PCR.
The workflow is:
\text{RNA} \rightarrow \text{cDNA} \rightarrow \text{PCR amplification}Two-step RT-PCR
In a two-step method:
- RNA is converted into cDNA in a separate reverse-transcription reaction.
- A portion of the cDNA is transferred to a conventional PCR reaction.
- The PCR product is analyzed, commonly by agarose gel electrophoresis.
This method is useful when the same cDNA sample will be tested against several different gene targets.
One-step RT-PCR
In one-step RT-PCR, reverse transcription and PCR occur sequentially in the same tube. This reduces handling and lowers the risk of contamination, but the resulting cDNA is generally not available for additional testing.
Main applications of RT-PCR
- Detection of RNA viruses.
- Analysis of messenger RNA.
- Gene-expression studies.
- Detection of RNA transcripts.
- Verification of RNA splicing.
- Molecular diagnostics.
- Analysis of RNA from cells and tissues.
Instruments used for RT-PCR
RT-PCR generally requires:
- A thermal cycler.
- A reverse-transcription system or thermocycler program.
- RNA extraction equipment or kits.
- RNase-free pipettes and consumables.
- Agarose gel electrophoresis equipment for endpoint detection.
The reverse-transcription reaction may be performed using a separate heat block, a small thermocycler, or the same conventional thermal cycler used for PCR.
What Is RT-qPCR?
RT-qPCR combines reverse transcription with quantitative real-time PCR. It is used to detect or quantify RNA while monitoring amplification through fluorescence.
The process is:
\text{RNA} \xrightarrow{\text{reverse transcriptase}} \text{cDNA}
\xrightarrow{\text{qPCR}} \text{quantified target}RT-qPCR can be performed in two ways:
- One-step RT-qPCR: Reverse transcription and qPCR occur in the same tube.
- Two-step RT-qPCR: cDNA is prepared first, followed by qPCR in a separate reaction.
RT-qPCR is commonly used for gene-expression analysis and RNA-virus detection.
Key Differences
| Feature | Conventional PCR | qPCR | RT-PCR | RT-qPCR |
|---|---|---|---|---|
| Full name | Polymerase chain reaction | Quantitative or real-time PCR | Reverse transcription PCR | Reverse transcription quantitative PCR |
| Starting template | DNA | DNA | RNA | RNA |
| Reverse transcription | No | No | Yes | Yes |
| Fluorescence monitoring | Usually no | Yes | Usually no | Yes |
| Result | Amplified DNA detected at the end | DNA quantified during amplification | cDNA/PCR product detected at the end | RNA target quantified through cDNA |
| Main instrument | Conventional thermal cycler | Real-time PCR system | Thermal cycler plus RT setup | Real-time RT-PCR instrument |
| Typical detection | Agarose gel or endpoint analysis | Fluorescence and Ct/Cq value | Agarose gel or endpoint analysis | Fluorescence and Ct/Cq value |
| Common use | DNA amplification | DNA quantification | RNA detection or cDNA generation | RNA quantification and diagnosis |
Instruments and Supporting Equipment
1. Conventional thermal cycler
A conventional thermal cycler is used for PCR and many endpoint RT-PCR procedures. It has a heated block that holds PCR tubes or a multiwell plate and changes temperature according to the programmed protocol.
It does not normally measure fluorescence during the reaction.
2. Real-time PCR instrument
A real-time PCR instrument performs thermal cycling and measures fluorescence after or during each cycle. It is required for qPCR and RT-qPCR.
The instrument’s optical system may detect several fluorescent channels, allowing multiplex testing with multiple targets in one reaction.
3. Microcentrifuge
A microcentrifuge is used to collect liquid at the bottom of tubes, separate phases during extraction, and briefly spin down PCR mixtures.
4. Micropipettes
Accurate micropipettes are essential for preparing master mixes, adding primers and probes, transferring nucleic acids, and setting up controls. Separate pipettes are recommended for:
- Reagent preparation.
- Sample addition.
- Amplified-product handling.
5. Nucleic-acid extraction equipment
RNA or DNA may be extracted manually using spin columns or with automated instruments. Examples of automated systems include:
- QIAGEN QIAcube.
- KingFisher Flex and KingFisher Apex.
- Roche MagNA Pure systems.
- QIAGEN EZ2.
- BioMérieux NucliSENS systems.
6. Agarose gel electrophoresis system
This is commonly used after conventional PCR or endpoint RT-PCR. It separates DNA fragments according to size and allows the amplified product to be visualized.
7. Biosafety cabinet
For clinical or potentially infectious specimens, sample preparation may require a certified biological safety cabinet. The exact biosafety requirements depend on the organism, specimen type, and institutional procedures.
Reagents Required
The exact reagents depend on the method, but typical components include:
Conventional PCR
- DNA template.
- Forward and reverse primers.
- Thermostable DNA polymerase.
- dNTPs.
- Magnesium ions.
- Reaction buffer.
- Nuclease-free water.
qPCR
- DNA template.
- Primers.
- Fluorescent dye or probe.
- qPCR master mix.
- DNA polymerase.
- dNTPs.
- Magnesium ions.
- Reaction buffer.
RT-PCR and RT-qPCR
- RNA template.
- Reverse transcriptase.
- DNA polymerase.
- Primers.
- dNTPs.
- Reaction buffer.
- RNase inhibitor, when required.
- Fluorescent dye or probe for RT-qPCR.
Controls Used in PCR Testing
Reliable PCR testing requires appropriate controls.
- No-template control: Contains all reaction components except the target nucleic acid. It helps identify contamination.
- Positive control: Contains a known target and confirms that the reaction works.
- Negative extraction control: Passes through the extraction procedure without a clinical sample and helps identify contamination during extraction.
- Internal control: Monitors extraction quality and possible PCR inhibition.
- No-RT control: Used in RT experiments to identify amplification caused by contaminating genomic DNA rather than RNA-derived cDNA.
Common Terminology Confusion
The terms qPCR and RT-PCR are sometimes used incorrectly.
- qPCR means quantitative real-time PCR and usually starts with DNA.
- RT-PCR means reverse transcription PCR and starts with RNA.
- RT-qPCR means reverse transcription followed by quantitative real-time PCR.
- Some laboratories use “real-time RT-PCR” or “rRT-PCR” to describe RT-qPCR for RNA targets.
The “RT” in RT-PCR refers to reverse transcription, not “real time.” Real-time measurement is indicated by “qPCR” or “real-time PCR.”
Simple Example
Suppose a laboratory wants to detect a DNA bacterium:
- Extract DNA from the specimen.
- Use conventional PCR to amplify a bacterial gene.
- Run the product on an agarose gel.
If the laboratory wants to measure the quantity of that bacterial DNA:
- Extract DNA.
- Perform qPCR with fluorescent primers or probes.
- Interpret the Cq value and standard curve.
If the target is an RNA virus:
- Extract RNA.
- Convert RNA to cDNA using reverse transcriptase.
- Amplify the cDNA using PCR or qPCR.
- For quantitative detection, use RT-qPCR.
Conclusion
Conventional PCR is mainly an endpoint DNA amplification method. qPCR is a real-time DNA quantification method that uses fluorescence. RT-PCR is designed for RNA templates, which are first converted into cDNA. RT-qPCR combines reverse transcription and real-time quantification and is especially useful for gene-expression studies and RNA-virus detection.
In short:
- PCR: DNA amplification.
- qPCR: Real-time DNA amplification and quantification.
- RT-PCR: RNA converted to cDNA and amplified.
- RT-qPCR: RNA converted to cDNA, then quantified by real-time PCR.